The regulation of cyclic 3',5' -nucleotide phosphodiesterase will be investigated utilizing a Ca2 ion -dependent phosphodiesterase from brain as a model system. This phosphodiesterase is deactivated by the removal on anion exchange chromatography of a Ca2 ion -dependent regulator (CDR) identified as a heat stable, acidic Ca2 ion -binding protein previously purified to homogeneity. The sensitive phosphodiesterase will be purified to homogeneity by conventional enzyme isolation techniques, stabilized, and characterized physically for amino acid composition and subunit structure. Interactions among Ca2 ion, CDR, cyclic nucleotide substrate, Mg2 ion, and the phosphodiesterase will be characterized by circular dichroic spectrophotometry, analyses of reaction kinetics, and direct binding studies. The molar ratio of CDR required for the activation of one mgle of enzyme will be determined. Antibodies will be prepared against the phosphodiesterase and used for establishing the subcellular localization of the enzyme by immunohistochemistry and for studying the protein turnover rate of the enzyme. Relationships between the Ca2 ion -dependent phosphodiesterase and various membrane-bound and cytoplasmic phosphodiesterases will be sought by physical comparisions on gel filtration chromatography, gel electrophoresis, and sucrose density ultracentrifugation, by comparison of reaction kinetics, and by cross reactivity of alternate phosphodiesterases with antibody prepared to the Ca2 ion -dependent enzyme. The reaction kinetics of a CDR requiring CA2 ion -moculated adenylate cyclase activity from brain will be characterized and the tissue distribution of CDR-dependent adenylate cyclase investigated. Functional relationships between the CDR requiring phosphodiesterase and the adenylate cyclase will be studied in a cloned cell line system in tissue culture, such as rat C-6 glioma cells, which possesses both activities. Insight gained in tissue culture systems will be later applied to understanding the physiological role of these enzymes in brain and other tissues.